Photocrosslinking GlcNAc (GlcNDAz)

GlcNDAz is an analog of GlcNAc in which the diazirine photocrosslinking is appended at the N-acyl position. The enzymes of the human GlcNAc salvage pathway cannot efficiently metabolize GlcNDAz. Therefore, to achieve metabolic incorporation of GlcNDAz into cellular glycoconjugates, we synthesize the 1-phospho version of GlcNDAz, and also stably transfect cells with a mutant (F383G) of the UDP-GlcNAc pyrophosphorylase 1 (UAP1) that is capable of converting GlcNDAz-1-P to UDP-GlcNDAz. The GlcNDAz-1-P compound that we use includes protecting groups on the phosphate and all hydroxyl groups. These protecting groups make the compound less polar, allowing it to readily enter mammalian cells. Once inside the cell, intracellular esterases catalyze protecting group removal. Human OGT can transfer GlcNDAz from UDP-GlcNDAz to serines and threonines of substrate proteins, resulting in the production of the O-GlcNDAz modification in place of the naturally occurring O-GlcNAc modification. Cells containing O-GlcNDAz can be subjected to UV irradiation, leading to covalent crosslinking between O-GlcNAcylated proteins and nearby molecules. These crosslinked complexes can be analyzed by immunoblot and mass spectrometry. For example, we used O-GlcNDAz crosslinking to demonstrate the proximity between O-GlcNAcylated FG-repeat nucleoporins and karyopherins, such as transportin-1. We do not yet know whether GlcNDAz is incorporated into other types of glycoconjugates, such as N-linked glycans or GalNAc-type O-linked glycans.